1. Field of the Invention
The invention relates to a new, flexible hyperbranched dendron having multiple binding sites for negatively charged nucleic acids or other polyanions (e.g., DNA), and the synthesis of the hyperbranched dendron. The complexes of hyperbranched polymers and DNA are less than 90 nm in diameter and are very stable for prolonged periods of time; and the polymer material is of low toxicity. The polymer/DNA complexes can bind to and transfect cells in the presence of any added negatively charged medium components (e.g., serum) with unexpectedly high efficiency. The in vitro transfection efficiency using the hyperbranched dendron of the invention is higher than transfection reagents that are currently commercially available. In addition, because of the high transfection efficiency and low cytotoxicity, the hyperbranched dendron shows good in vivo transfection efficiency and includes lasting gene expression.
2. Introduction
Non-viral gene transfer is frequently regarded as a potentially more safe alternative to the viral gene delivery. However, non-viral gene transfer to mammalian cells usually lacks the efficiency typical to that of viral transduction and requires substantial improvement. Non-viral delivery of DNA usually requires condensation with positively charged lipids or polycations to enable binding of polyionic complexes to plasma membrane and further internalization by cells. The condensation ideally yields complexes that could release DNA after the internalization. In recent years several cationic polymers that readily form complexes with DNA (e.g. polylysine, polyethyleneimine or various types of block and graft copolymers) have been investigated as potential nonviral vectors that enable transfer of DNA into mammalian cells. Interactions of a polycation and DNA frequently results in a formation of very compact colloidal complexes which are frequently unstable and may precipitate from solutions [Pelta, J., et al., “DNA aggregation induced by polyamines and cobalthexamine,” J Biol Chem. 271, 5656–5662 (1996).]. Physical properties of these complexes that define their size and stability depend heavily on chemical structure as well as on physical properties of the polycation.
As research in the area of synthetic carriers for DNA delivery expands beyond the traditional in vitro experiments, various polycations, including branched polyethyleneimine (BPEI), starburst polyamidoamine (PAMAM) dendrimers [Haensler, J., et al., “Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture,” Bioconjugate Chem. 4, 372–379 (1993); Ohsaki, M., et al., “In vitro gene Transfection using dendritic poly(L-lysine),” Bioconjugate Chem. 13, 510–517 (2002); KukowskaLatallo, J. F., et al., “Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers,” Proc. Natl. Acad. Sci. U.S.A. 93, 4897–4902 (1996); Choi, J. S., et al., “Synthesis of a barbell-like triblock copolymer, poly(L-lysine) dendrimer-block-poly(ethylene glycol)-block-poly(L-lysine) dendrimer, and its self-assembly with plasmid DNA,” J. Am. Chem. Soc. 122, 474–480 (2000).], as well as hyperbranched polymers are now being closely investigated.
Some of the above polycations have shown high levels of gene transfer in mammalian cell culture. High transfection efficiency of BPEI and PAMAM compared to other polycations is explained by the effect of a “proton sponge” [Boussif, O., et al., “Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold,” Gene Ther. 3, 1074–1080 (1996)]. This effect is supposedly caused by the protonation of tertiary amines present in the core of those polymers in acidic milieu of endosomes. Godbey et al. [“Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery,” Proc. Natl. Acad. Sci. U.S.A. 96, 5177–5181 (1999)] reported that the protonated form of branched PEI or PAMAM may perturb endosomes probably due to membrane activity of cationic polymers or by osmotic effects causing occasional lysis. Haensler et al. (supra) has reported that dendrimers show high level of transfection in a wide variety of cells in culture with low concomitant cytotoxicity. However, in a later study they reported that stringently synthesized and purified, monodisperse PAMAM dendrimers showed low levels of transfection.
Furthermore, the transfection activity of PAMAM dramatically improved after the random degradation by heat treatment in a solvolytic solvent [Tang, M. X., et al., “In vitro gene delivery by degraded polyamidoamine dendrimers,” Bioconjugate Chem. 7, 703–714 (1996)] leading to a hypothesis that polydisperse “fractured” dendrimers are more flexible than monodisperse PAMAM enabling “fractured” dendrimer to form compact complex with DNA. Interestingly, fractured dendrimers are able to swell when released from DNA. Recently, Lim et al. [Lim, Y. B., et al., “Cationic hyperbranched poly(amino ester): A novel class of DNA condensing molecule with cationic surface, biodegradable three-dimensional structure, and tertiary amine groups in the interior.” J. Am. Chem. Soc. 123, 2460–2461 (2001); Lim, Y. B., et al., “Biodegradable, endosome disruptive, and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier,” Bioconjugate Chem. 13, 952–957 (2002)] reported biodegradable hyperbranched poly(amino esters) with high transfection efficiency and low cytotoxicity in vitro. This polymer is attractive, because unlike PAMAM dendrimer, it degrades within several days at physiological pH and, in addition, can be synthesized more easily than the fractured dendrimer.
The majority of PEI-mediated transfections use a low-branched polymer having MW from 25 kD to 800 kD that show good transfection efficiency in vitro [Boussif, O., et al., “Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold,” Gene Ther. 3, 1074–1080 (1996); Fischer, D., et al., “A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity,” Pharm. Res. 16, 1273–1279 (1999); Godbey, W. T., et al., “Poly(ethylenimine) and its role in gene delivery” J. Control. Release. 60, 149–160 (1999)]. Unfortunately, high molecular weight PEI has high cytotoxicity that limits its scope of potential applications in vivo. To decrease the cytotoxicity and to increase water solubility of the DNA polymer complex, polyethylene glycol-graft-PEI has been obtained. Grafted PEI has lower transfection efficiency than the non-grafted PEI but the latter drawback is compensated with a low cytotoxicity at high N/P ratio. There appears to be only one published report by Fischer, et al., (supra) describing the synthesis of a low molecular weight non-toxic branched PEI that showed at least two orders of magnitude higher transfection efficiency than a commercially available high molecular weight PEI. However, it is known that despite having a very low cytotoxicity, linear PEI (LPEI) is a less efficient transfection reagent than branched 25K PEI. As tertiary and secondary amine groups appear to cause less toxic effects than primary amine, LPEI is less toxic than branched low molecular weight PEI due to prevalence of secondary amino group. Overall, it appears that LPEI could be a good candidate for in-vitro and in-vivo gene delivery [Ferrari, S., et al., “ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo,” Gene Ther. 4, 1100–1106 (1997); Goula, D., et al., “Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system,” Gene Ther. 5,712–717 (1998); Goula, D., et al., “Polyethylenimine-based intravenous delivery of transgenes to mouse lung,” Gene Ther. 5, 1291–1295 (1998)].
However, none of the previously described non-toxic polycations appear to form sufficiently stable submicron complexes with DNA that could be useful for gene transfer in the presence of serum. The ability to preserve transfection ability in serum is critical for in vivo gene delivery. Therefore, the goals of the present invention include designing a high transfection efficiency polymer system that: 1) would form nanosized, stable complexes with plasmid DNA that would be amenable to a long-term storage; 2) would be efficient at low N/P ratios in presence of serum; 3) would have low cytotoxicity and/or 4) would be useful for in vivo gene delivery.